*2.1. Parasites*

Trypanosome isolates used in this study were the *T*. *cruzi* clone CL Brener (CLB) (TRYCC426, [12], and G strain [13]), *T*. *cruzi marinkellei* (TCC344)*, T. rangeli* SC58 [14] and *T*. *brucei rhodesiense* YTAT 1.1. The epimastigotes of *T*. *cruzi*, *T. cruzi marinkellei*, and *T. rangeli* were grown in axenic cultures at 28 ◦C in liver-infusion tryptose (LIT) medium [15] supplemented with 10–20% heat-inactivated fetal calf serum. Procyclic forms of *T*. *brucei rhodesiense* YTAT 1.1 were cultured in a semi-defined medium (SDM-79) supplemented with 10% heat-inactivated fetal bovine serum at 27 ◦C. *T. cruzi* extracellular amastigotes were obtained by culture tissue trypomastigote differentiation in a LIT medium, as previously described [16].

## *2.2. Identification of RHS Sequences in T. cruzi and T. cruzi marinkellei Genome Databases*

The search for homologous RHS genes in the TriTrypDB and GenBank databases was performed using the algorithms BLASTp, tBLASTn, BLASTx, and the presence of RHS domain architecture was confirmed using rpsBLAST [17]. RHS transcripts of CLB were used as queries to identify homologous sequences in other *Trypanosoma* species using the tBLASTn (e-value of 1 × 10−<sup>3</sup> ) search program. The retrieved sequences were evaluated for the presence of RHS domains with the rpsBLAST algorithm

(e-value of 1 × 10−<sup>5</sup> ) against the database of conserved domains [18]. An extra round of tBLASTn was performed using found RHS sequences as a query to improve genome survey sensibility. Figure S1 shows the flowchart of this analysis. Sequence alignments were carried out with RHS of clone CLB excluding truncated sequences. The nucleotide and amino acid sequences were aligned using the MUSCLE program [19] and the poorly conserved regions were removed using the Gblocks program [20].

### *2.3. Classification and Phylogenetic Analyses of RHS*

For these analyses, we selected RHS transcripts of the *T. cruzi* clone CLB [21]. Transcribed genes were analyzed for the presence of RHS domains with the rpsBLAST algorithm using 1 × 10−<sup>5</sup> e-value against the NCBI Conserved Domain Database (CDD) [18] (Figure S1). Sequences that showed false-positive RHS domains and pseudogenes were excluded. In the phylogenetic analysis, the global multiple alignment was carried out with the MUSCLE algorithm [19]. Phylogenetic trees were generated using the "Maximum likelihood method" using the RaxML v 8.2.9 program [22], with an automatic search for substitution models (PROTGAMMAAUTO) selected by the Akaike information criterion (AIC) (auto-prot = AIC) information criterion, with 1000 bootstrap replicas. The phylogenetic tree was visualized with the program FigTree V 1.4.2 [23].
